Bioremediation of Chromium and Arsenic from Industrial Wastewater Michael J. McInerney and Lee Krumholz, University of Oklahoma Shahida Hasnain and Muhammad Faisal, University of the Punjab, LahorePakistani Funding (HEC): $ 268,000US Funding (USAID): $ 237,460Project Dates on US Side: April 1, 2008 - November 30, 2011

Project Overview

The continuous industrial usage of chromium (Cr) and arsenic (As) compounds by different industries has polluted the aquatic and terrestrial environment in Pakistan. Microbial processes in soils and sediments devoid of air could act to increase or decrease health risks associated with these metal depending on which microbial process becomes dominant. In soils and sediments devoid of air, reductive microbial processes will increase the health risk due to arsenic contamination by reducing the less toxic and less mobile form of As (arsenate, AsV) into a more toxic and mobile form of arsenic (arsenite, ArIII). On the other hand, reductive microbial process will decrease the health risk due to chromium contamination by reducing the more toxic and more mobile form of Cr (CrVI) into a less toxic and less mobile form of chromium (CrIII). Thus, it is essential to know what microbes are present in these contaminated soils in order to assess health risk and devise appropriate mitigation strategies. The objectives of the project are as follows:

To isolate and characterize chromium and arsenic resistant bacteria from contaminated Pakistani soils and evaluate their potential to

Detoxify chromium by converting CrVI to CrIII

Detoxify arsenic by converting ArIII to ArV

Increase arsenic toxicity by converting ArV to ArIII

Leather Tanning Site

To determine how metal contamination, soil chemistry and geography influences the microbial community in order to predict the fate of arsenic and chromium in contaminated soil and water.

To train new Pakistani scientists in anaerobic microbiology, molecular ecology, and environmental genomics

Major Results

Isolated and characterized microbes highly resistant to metals that can be used in cleaning up contaminated soils

Developed plant cultivars able to grow in soils highly contaminated with metals that can be used for phytoremediation and food production

Trained more than one hundred Pakistani students in ecological genomics and anaerobic microbiology via distance learning

Hosted two Pakistani PhD students for extended visits to the University of Oklahoma to learn about advanced environmental microbiology techniques

Discovered diverse gene sequences involved in arsenic resistance

Quarterly Update

During the final stage of the project in 2011, arsenate oxidase and chromate reductases enzymes are being purified and characterized, and Pakistani scientists are testing these enzymes for their ability to detoxify toxic arsenic and chromium. The OU researchers will be analyzing additional samples from Kasur, Sheikhupura, and Sialkot to quantify the number of 16S rRNA gene copies at each site using quantitative PCR and determine what effect chromium and arsenic exposure have on the functionality of the communities by using GeoChip. The project team also plans to use primers for the arsB gene and the ACR genes to obtain PCR products so that they can examine the differences in arsenic gene communities present in the soil samples. The stoichiometry of electron donor and electron acceptor use by isolates that reduce arsenate and oxidize asenite is expected to be determined; new enrichments will be established; and the diversity present in enrichments that transform sulfur compounds will also be characterized. Supported by university endowment funds provided by the US PI, Mr. Yasir Rehman, a visiting PhD student from University of the Punjab, will be able to extend his visit at OU through the end of October 2011, which will give him a full year of microbial ecology training in Dr. McInerney’s lab. Although funding under this project is scheduled to end as November 30, another Pakistani scholar from University of the Punjab, Ms. Fariha Rizvi, recently received a fellowship from HEC to study in Dr. McInerney’s laboratory for 6 months. At last report she was expected to start her visit in September 2011, pending visa issuance, so the linkages established under this project will continue even after the grant ends.

After Dr. Hasnain's lab received several key pieces of new equipment in January 2010, they began anaerobic microbiology work applying the training provided by their OU colleagues. In 2010, Dr. Hasnain and her students intensively sampled another contaminated area and extracted DNA from the samples for subsequent analysis at OU. Oklahoma researchers also have made several advances toward characterizing the microbial flora in chromium and arsenic contaminated soils. Using DNA isolated by the Pakistani researchers from soils contaminated with arsenic and chromium, they were able to construct barcoded PCR libraries using universal 16S rRNA gene primers. The Oklahoma research team also focused on finding primers that are specific enough to detect the arsenic resistance genes in isolates and general (degenerate) enough to detect these genes in soil DNA; and success was finally attained with two sets of primers.

Regarding the fourth objective of this project, two Pakistani PhD students arrived at OU in the fall of 2010 for six months of training in anaerobic microbiology and molecular ecology. Meanwhile, Dr. McInerney and his colleagues have continued to provide data analysis training to other Pakistani students via distance learning. At present, this project is supporting three PhD students at University of Punjab, Lahore and one PhD student and two MS students at the University of Oklahoma. Dr. Hasnain and her co-PI attended the 6th International Conference on Plant Tissue Culture and Biotechnology at Dhaka University, Dhaka Bangladesh and presented the project in December 2010. She and Dr. McInerney had another opportunity for in-person consultations when they participated in the Pakistan-US Science and Technology Conference in Dubai in March 2011.

Although the Oklahoma researchers have been unable to provide training in Lahore in person due to the security situation, they gave produced a series of six video-based modules, the first five of which were provided to the Pakistani counterparts via YouTube in June 2009 to train them in various aspects of anaerobic microbiology lab procedures, including (1) construction of a gassing station, (2) media preparation, (3) transfer of cultures, (4) exchange and pressurization of the gas phase, and (5) preparation of microcosms. A two-part sixth video on extracting DNA from soils was made available in February 2010. They have also provided the Pakistani researchers with written protocols and suggestions for DNA extraction from soils, and additional training on these procedures was provided to Dr. Shahida Hasnain when she visited OU in December 2009. Regular videoconferences are being held to facilitate training and ongoing research collaboration.

During the first year of the project, which began on the US side in April 2008, the Pakistani researchers collected contaminated and uncontaminated soil samples near tanneries from three sites in Din Garh (Kasur) and East Pakistan. They isolated eight bacteria resistant to Cr(VI) and eight bacteria resistant to Ar(III). Extensive phenotypic characterization of all 16 stains was completed, and the US partners assisted their colleagues with genetic characterization of these strains. (In 2009, 45 more strains highly resistant to arsenic were collected from additional sites in Shaikhupura and Silakot, and they have been characterized for growth and metabolic properties.) The Pakistani research team is also working to characterize the strains for their ability to reduce Cr(VI) and oxidize As(III), and the US side has been providing training in the analytical methods needed to quantify the metals in various oxidation states. In order to test Pakistani soil samples in the United States, the US researchers first had to secure the necessary import permits as well as space in a lab facility approved for work with foreign soils. With these hurdles overcome, they received the first batch of soil samples in November 2008 and began extracting and sequencing DNA from bacteria in the soil. Unfortunately, however, they found that the samples had deteriorated during shipment, so they decided instead to train the Pakistanis on how to extract DNA in their own lab in Lahore, as the DNA samples could easily be shipped without damage.